In certain circumstances, the immune system must be controlled in order to either augment a deficient response or suppress an excessive response. For example, when organs such as kidney, heart, lung, bone marrow, and liver are transplanted in humans, the body will sometimes reject the transplanted tissue by a process referred to as allograft rejection. In treating allograft rejection, the immune system is frequently suppressed in a controlled manner through drug therapy with immunosuppressant drugs including, but not limited to, cyclosporin, tacrolimus, sirolimus, mycophenolic acid, and everolimus. Immunosuppressant drugs are carefully administered to transplant recipients in order to help prevent allograft rejection of the foreign (i.e. non-self) tissue. Many of the immunosuppressant drugs require the measurement of their concentrations in blood with subsequent dosage adjustment to maximize efficacy while minimizing toxicity. Thus, regular monitoring of immunosuppressant drug blood levels is an essential component of the post transplant medical regimen.
Traditionally, immunosuppressant drug monitoring has been performed using immunoassays. However, there exist some known immunosuppressant drug assays which employ tandem mass spectrometry coupled to liquid chromatography, the technique sometimes known as LC-MS/MS. Generally speaking, (LC-MS/MS) is an extremely useful technique for detection, identification and (or) quantification of components of mixtures or of analytes within mixtures. Specifically, this technique has further been found to be a powerful analytical tool that provides high specificity and sensitivity in measurement of immunosuppressant drugs. Because of a relatively simple sample preparation procedure and high sensitivity and specificity, LC-MS/MS has shown great potential to be the method of choice for measuring immunosuppressant drugs, especially for simultaneous multiple-drug monitoring.
In general, liquid chromatography coupled with mass spectrometry (the general technique known as LC-MS) provides data in the form of a mass chromatogram, in which detected ion intensity (a measure of the number of detected ions) as measured by a mass spectrometer is given as a function of time. In the LC-MS technique, various separated chemical constituents elute from a chromatographic column as a function of time. As the various constituents are eluted from the column, they are submitted for mass analysis by a mass spectrometer. The mass spectrometer accordingly generates, in real time, detected relative ion abundance data for ions produced from each eluting analyte, in turn.
Mass spectrometry (MS) is an analytical technique to filter, detect, identify and/or measure compounds by the mass-to-charge ratios of ions formed from the compounds. The quantity of mass-to-charge ratio is commonly denoted by the symbol “m/z” in which “m” is ionic mass in units of Daltons and “z” is ionic charge in units of elementary charge, e. Thus, mass-to-charge ratios are appropriately measured in units of “Da/e”. Tandem mass spectrometry (MS/MS) techniques generally include (1) ionization of compounds to produce ion species of different respective m/z ratios; (2) selection and isolation of one (or a few) specific ion species; (3) fragmentation of the selected ion species so as to generate product ions; and (4) detection and analysis of the mass-to-charge ratios of particular diagnostic fragment ion species; and determination of analyte abundances in the original sample from measured quantities of the diagnostic fragment ion species.
Immunosuppressant drugs (ISDs) are often analyzed in whole-blood using LC-MS or LC-MS/MS. For example, one known quantitative assay for the research-use only determination of the ISD compounds Cyclosporin A, Sirolimus, Tacrolimus and Everolimus in whole blood specimens is provided by the ClinSpec™ Immunosuppressants Test kit provided by Thermo Scientific of Waltham Mass. USA. Sample preparation is based on simple protein precipitation. Whole blood samples are treated with a provided extraction solution to precipitate protein and extract the compounds of interest into the organic phase. The supernatant from the protein precipitated whole blood sample is then injected onto a column provided with the kit. The analyte(s) of interest as well as provided internal standards are then eluted into the ionization source of the mass spectrometer.
The chromatographic analytical column used in this known ClinSpec™ assay is a C8 reverse-phase high-performance liquid chromatography (HPLC) column (specifically a 10 mm long Thermo Scientific Javelin™ guard column packed with Thermo Scientific Hypersil Gold™ 5 μm particles. An atmospheric pressure chemical ionization (APCI) ion source is used in positive-ion mode in the mass spectrometer. The precursor ion species for Tacrolimus, Sirolimus and Cyclosporin A and Everolimus are at m/z ratios of 821.4, 931.6, 1219.9 and 975.7, respectively. The monitored product ions formed by fragmentation of these parent ion species are at m/z ratios of 768.3, 864.5, 1202.9 and 908.4, respectively. Ascomycin and Cyclosporin D are used as internal standards.
Although the above-described ISD assay utilizes an APCI source, the use of heated electrospray (H-ESI) ionization could improve the sensitivity of the assay. Further, many analytical or clinical laboratories are set up to routinely perform LC-MS analyses using electrospray ionization and would suffer inconvenience or decreased efficiency from having to re-configure and or re-calibrate their systems for APCI. Unfortunately, a difficulty arises in the use of electrospray ionization in conjunction with mass spectrometry of ISDs from clinical samples. This difficulty arises from the co-elution of phospholipids. Since the phospholipids have similar hydrophobicity characteristics to the ISD analyte compounds, they are chromatographically separated only with difficulty from the analytes. The problem when using electrospray ionization is that the co-eluting phospholipids causes strong ion suppression of the analyte compounds. The general phenomenon of ion suppression during electrospray ionization has been shown (King R, et al., “Mechanistic investigation of ionization suppression in electrospray ionization”, J Am Soc Mass Spectrom 2000; 11:942-50) to result from a change in the efficiency of droplet formation or droplet evaporation in the presence of the non-volatile or less volatile interfering compound, thereby reducing the quantity of analyte ions that are formed.
The conventional approach to eliminating the problem of ion suppression of ISDs in the presence of co-eluting phospholipids would be to improve the chromatographic separation of these two types of compounds, either by installing a second “cleanup” column upstream from the analytical column or by increasing the length of the analytical column. However, the inventors have discovered that installing a commonly-used TurboFlow™ High-Turbulence Liquid Chromatography cleanup column does not sufficiently separate phospholipids from ISD compounds as a result of the similar hydrophobicities of these two types of compounds. The inventors have also discovered that, somewhat surprisingly, increasing the length of the analytical column from its conventional length of 50 mm in fact exacerbates the problem of ion suppression by co-eluting phospholipids.
Accordingly, there is a need in the art for improved techniques—both apparatus and methods—for performing LC-MS assays of immunosuppressant drugs in patient samples using electrospray ionization. Such methods must eliminate ion suppression from unwanted matrix components such as phospholipids. Although stable isotopes for each ISD are available to compensate for interferences, it is best to minimize such interferences, in order to maintain analysis accuracy at low concentration levels and to maintain a reliable lower limit of quantitation of these compounds.